Erucin, an H2S-Releasing Isothiocyanate, Exerts Anticancer Effects in Human Triple-Negative Breast Cancer Cells Triggering Autophagy-Dependent Apoptotic Cell Death

Breast cancer is the most frequent form of cancer occurring in women of any age. Among the different types, the triple-negative breast cancer (TNBC) subtype is recognized as the most severe form, being associated with the highest mortality rate. Currently, there are no effective treatments for TNBC. For this reason, the research of novel therapeutics is urgently needed. Natural products and their analogs have historically made a major contribution to pharmacotherapy and the treatment of various human diseases, including cancer. In this study, we explored the potential anti-cancer effects of erucin, the most abundant H2S-releasing isothiocyanate present in arugula (Eruca sativa) in MDA-MB-231 cells, a validated in vitro model of TNBC. We found that erucin, in a concentration-dependent manner, significantly inhibited MDA-MB-231 cell proliferation by inducing apoptosis and autophagy. Additionally, erucin prevented intracellular ROS generation promoting the expression of key antioxidant genes and halted MDA-MB-231 cell migration, invasion, and colony formation. In conclusion, using a cellular and molecular biology approach, we show that the consumption of erucin could represent a novel and promising strategy for intervention against TNBC.


Introduction
Triple-negative breast cancer (TNBC) accounts for about 12-20% of all breast cancer cases and affects with higher frequency young women carrying BRCA1 gene expression [1]. The term triple-negative refers to the fact that breast cancer cells lose the expression of three major classes of hormone receptors: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). These alterations make patients refractory to conventional hormonal therapies, causing major difficulties in clinical decision making and management. Current treatment options are limited to surgery, adjuvant chemotherapy, and radiotherapy. Although progress has been made over the last century in cancer research, TNBC still causes the highest rate of deaths among all types of breast cancer [2]. For this reason, there is an urgent need to discover and develop new therapeutics to treat effectively TNBC. In this regard, numerous therapeutic dietary or non-dietary compounds present in plants have gained much interest due to their preventive and protective action against different types of cancer [3][4][5]. A considerable number of pre-clinical and clinical studies show that phytochemicals exert protective effects against the growth and spread of tumor cells by regulating their proliferative capacity, and survival, as well as counteracting the production of reactive oxygen species (ROS) [6,7]. Among edible phytochemicals, the consumption of cruciferous vegetables belonging to the large family of Brassicaceae (also called Cruciferae) has been reported to decrease the risk of developing several types of cancers, including lung, colorectal, ovarian, pancreatic, and breast cancer [8][9][10]. The Brassicaceae family includes the rocket plant, which is an annual plant very popular all around the world for the edible green leaves commonly used for salad and food dish recipes. The two main varieties of rocket plants are Diplotaxis tenuifolia (wild rocket) and Eruca sativa (arugula, or cultivated rocket). Eruca sativa contains a wide range of compounds with nutraceutical and organoleptic properties, including isothiocyanates, phenolic compounds, and carotenoids. Among them, erucin (4-methylthiobutyl isothiocyanate) is one of the major components of leaves obtained from the enzymatic hydrolysis of glucoerucin. As demonstrated in a large number of in vitro and in vivo studies, erucin exerts protective effects against several types of human cancers, including breast cancer [8,[11][12][13][14][15]. Remarkably, recent findings revealed that the protective effect of erucin relies on its intrinsic capacity to slowly release hydrogen sulfide (H 2 S), which is one of three gasotransmitters, along with nitric oxide (NO) and carbon monoxide (CO), which act in our body to modulate numerous biological processes [16].
Despite that, the potential use of erucin against TNBC has not yet been investigated. Toward this goal, in this study we explored the potential anticancer effect of erucin on cell proliferation and survival, ROS production, migration, and invasion in human MDA-MB-231 cells using an in vitro model of TNBC.

Erucin Inhibits Proliferation and Induces Apoptosis in Human TNBC Cells
To investigate the potential effect of erucin in human TNBC cells, we measured the proliferation capacity of MDA-MB-231 cells exposed to a crescent concentration of erucin using the MTT assay. As shown in Figure 1A, erucin in a concentration-dependent manner (1-100 µM) inhibited the growth of MDA-MB-231 cells after 48 h with an IC50 of about 24 µM. Subsequently, we explored the potential effects of erucin on cell necrosis and apoptosis in the same cell line using FACS analysis. Using this approach, we found that erucin (30 µM) induced both early and late cell apoptosis in about 60% of MDA-MB-231 cells ( Figure 1B). This effect was then confirmed by western blot analysis, revealing the time-dependent cleavage (hence activation) of Caspase-3 and its substrate poly (adenosine diphosphateribose) polymerase (PARP) by erucin ( Figure 1D).

Erucin Promotes Autophagy in Human TNBC Cells
In the next stage, we explored whether erucin could modulate autophagy in MDA-MB-231 cell; this is an evolutionarily conserved cellular process that is importantly intertwined with apoptosis and plays a critical role in regulating cancer-cell development and proliferation [17]. We found that erucin (30 µM), again in a time-dependent manner, promoted autophagy in MDA-MB-231 cells; this was reflected by the increased expression of key regulatory genes, including ULK1, ATG13, BECN1, and BNIP3 (Figure 2A-D). Additionally, by western blot analysis, we found that erucin significantly increased the expression of LC3II protein, which is the lipidated form of the LC3I ( Figure 2F,G), and concomitantly decreased the expression of p62. These latter are known to be two key positive and negative regulators of autophagosome formation and activity, respectively ( Figure 2F-H).

Erucin Reduces ROS Production by Inducing the Expression of Antioxidant Genes in Human TNBC Cells
Cumulative evidence shows that the unbalanced formation of reactive oxygen (ROS) species is one of the major pathological mechanisms underpinning cancer development and progression [18]. In light of this, a key step in this study was to understand if the protective effect of erucin observed in MDA-MB-231 cells could also be ascribed to the formation of ROS species. Toward this goal, MDA-MB-231 cells were exposed to H 2 O 2 /Fe 2+ (2 mM), which is an efficient method to increase the production of ROS in in vitro cell model systems. We found that pre-treatment for 3 and 6 h with erucin (30 µM) in MDA-MB-231 cells exposed to H 2 O 2 /Fe 2+ significantly prevented ROS formation as determined by the DCF fluorescence intensity ( Figure 3A,B). To investigate the mechanism through which erucin was capable of preventing the ROS formation in TNBC cells, we measured the expression of key genes encoding for antioxidant enzymes, including heme oxygenase-1 (HMOX-1), Glutamate-Cysteine Ligase Catalytic Subunit (GCLC), Glutamate-Cysteine Ligase Modifier Subunit (GCLM), and Superoxide Dismutase 1 (SOD1). Treatment with erucin 30 µM for 3 and 6 h significantly increased the expression of all the aforementioned genes in a time-dependent manner ( Figure 3C,D). In addition, we found that erucin induced the translocation from the cytoplasm to the nucleus of NF-E2-related factor 2 (Nrf2), which is one of the best-known transcription factors regulating the expression of numerous antioxidant genes; it is also concomitantly reduces the expression of its repressor Kelch-like ECH-associated protein 1 (Keap1) ( Figure 3E,F).

Erucin Promotes Autophagy in Human TNBC Cells
In the next stage, we explored whether erucin could modulate autophagy in M

Erucin Reduces ROS Production by Inducing the Expression of Antioxidant Genes in Human TNBC Cells
Cumulative evidence shows that the unbalanced formation of reactive oxygen (ROS) species is one of the major pathological mechanisms underpinning cancer development and progression [18]. In light of this, a key step in this study was to understand if the protective effect of erucin observed in MDA-MB-231 cells could also be ascribed to the formation of ROS species. Toward this goal, MDA-MB-231 cells were exposed to H2O2/Fe 2+ (2 mM), which is an efficient method to increase the production of ROS in in vitro cell model systems. We found that pre-treatment for 3 and 6 h with erucin (30 µM) in MDA-MB-231 cells exposed to H2O2/Fe 2+ significantly prevented ROS formation as determined by the DCF fluorescence intensity ( Figure 3A,B). To investigate the mechanism through which erucin was capable of preventing the ROS formation in TNBC cells, we measured the expression of key genes encoding for antioxidant enzymes, including heme oxygenase-1 (HMOX-1), Glutamate-Cysteine Ligase Catalytic Subunit (GCLC), Glutamate-Cysteine Ligase Modifier Subunit (GCLM), and Superoxide Dismutase 1 (SOD1). Treatment with erucin 30 µM for 3 and 6 h significantly increased the expression of all the aforementioned genes in a time-dependent manner ( Figure 3C,D). In addition, we found that erucin induced the translocation from the cytoplasm to the nucleus of NF-E2-related factor 2 (Nrf2), which is one of the best-known transcription factors regulating the expression of numerous antioxidant genes; it is also concomitantly reduces the expression of its repressor Kelch-like ECH-associated protein 1 (Keap1) (Figure 3 E,F).

Discussion
Natural compounds, including those present in Cruciferae, are shown to significantly prevent and decrease cancer development and progression. In particular, the protective effect of Cruciferae is attributed to isothiocyanates, a large class of products derived by the hydrolysis of glucosinolates, including sulforaphane and erucin [19]. Remarkably, in the last few years, the potential use of erucin as a new strategy to tackle chemoresistance in cancer emerged owing to its ability to remove oxidative stress and halt inflammatory signaling [20][21][22]. In a recent review article, Singh and colleagues report that erucin exerts anticancer effects by inhibiting carcinogen-activating enzymes and promoting the activity of detoxifying enzymes and the mechanisms leading to the inhibition of metastasic processes and angiogenesis [23]. Additionally, erucin leads to the activation of apoptotic processes in cancer cells by inducing cell cycle arrest and suppressing microtubule dynamics [24,25]. However, erucin potentiates the anticancer activity of 4-hydroxytamoxifen in estrogen receptor-positive breast cancer cell lines [26] and, in combination with lapatinib, seems to overcome drug resistance in the same cells [27,28].
H 2 S donors and their derivatives were demonstrated to effectively reduce the proliferation and apoptosis of TNBC cells by inhibiting the phosphorylation or expression of proteins associated with NF-κB, PI3K/Akt/mTOR and Ras/Raf/MEK/ERK signaling pathways. They also inhibited the expression of MMP-2/9 and EMT to resist TNBC metastasis by inhibiting aberrant activation of the β-catenin pathway [29]. Recently, Citi et al. reported that erucin limits the proliferation, growth, and survival of pancreatic cancer cells through a mechanism dependent on the slow release of H 2 S [13]. However, the role of H 2 S in cancer is still matter of debate since, in the current relevant literature, there are studies either supporting a beneficial or detrimental role on cancer development, differentiation, metastasis, and proliferation. These divergent effects are most likely due to different effect in different type of cancers as well as the methodological approaches used. Another variant to be taken into account is the release kinetic of H 2 S. Indeed, H 2 S is known to produce hormetic responses, which refer to a biphasic dose/concentration effect. Typically, a low dose produces stimulatory or beneficial effects, while a high dose produces inhibitory or toxic effects [30][31][32]. The second important issue is how much of the hydrogen sulfide released effectively penetrates the cells. Erucin it has been shown to release H 2 S inside cells in a dose-dependent manner [33]. In our experimental settings, erucin reduced proliferation in a concentration-dependent manner of MDA-MB-231 cells, which is an elective model of TNBC with an high proliferative capacity. In particular, at the concentration selected 30 µM erucin causes an inhibition of cell proliferation of about 50%. Moreover, at this concentration erucin also promotes a significant apoptosis concomitantly enhancing the expression of key genes and proteins (BECN1, LC3, ULK1, ATG13, BNIP3) triggering and driving autophagy. Thus, erucin acts on both autophagy and apoptosis, with these functions being intimately intertwined to control, among numerous processes, the cell fate [34,35]; this approach represents a promising target in many types of cancer. Intracellular imbalance in ROS production is one of the major pathological mechanisms underpinning tumor initiation and metastatic progression [36]. Treatment of cells with erucin significantly prevented the intracellular formation of ROS in TNBC cells. This effect could be simply due to the ability of the H 2 S released intracellularly by erucin to scavenge ROS directly [37]. However, based on our results, the antioxidant effect of erucin is not only exerted through the direct "quenching" effect of H 2 S on ROS; erucin also induces the expression of Nrf2 and concomitantly reduces the expression of its repressor Keap1. Nrf2 is also a key transcriptional factor acting to promote the expression of numerous antioxidant genes, including GCLC, GCLM, SOD1, and HMOX-1. Since the expression of all the aforementioned genes was increased in MDA-MB-231 cells, erucin displays a direct quenching effect coupled to a molecular modulation on Nrf2, as shown by other researchers [38]. To further characterize the erucin profile, we studied the ability of this H 2 S donor to interfere in vitro with migration, invasion, and colony formation of MDA-MB-231 cells. To perform this study, we used doses that do not trigger apoptosis and/or autophagy. Erucin, at the 1 µM concentration, showed a significant inhibitory effect on migration. The same dose caused an even more marked inhibitory effect on invasion and colony formation, giving about 80% on both parameters evaluated. These results further support the proposal that erucin effects involve a reduction in both ROS and more than one molecular mechanism.
In conclusion, our study provides novel insights into the use of hydrogen sulfide donors as a promising strategy for curative and/or adjuvant therapies against TNBC. Moreover, our data suggest that low and slowly delivered levels of hydrogen sulfide intracellularly are necessary to achieve beneficial effect. However, future studies are necessary to explore the effect of erucin alone or in combination with conventional anticancer agents using in vivo models of TNBC.

Western Blot Analysis
Total proteins were obtained from cells using a lysis RIPA buffer (50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, and 0.25% (v/v) sodium deoxycholate, 0.1% SDS) supplemented with Protease Inhibitor Cocktail (cat. P8340-1 mL, Merk, Milan, Italy). Protein lysates were quantified by the Bradford method (cat. 5000006, Bio-Rad, Italy). An equal amount of proteins (40 µg) from each cell extract was loaded and separated on sodium dodecyl sulphate polyacrylamide (SDS-PAGE) gel; it was then transferred on a nitrocellulose membrane using Trans-Blot Turbo Transfer Starter System (Bio-Rad, Italy). After transfer, the membranes were blocked in 5% low-fat milk in PBS with 0.1% Tween 20 (PBST) for 1 h at room temperature before being incubated overnight at 4 • C with the following primary antibodies: sc-47,778; Santa Cruz Biotechnology, Santa Cruz, CA; diluted 1:1000). β-actin and α-tubulin were used as the control normalizing protein for cytosolic and nuclear lysates, respectively. The membranes were washed three times with PBST and incubated with anti-mouse (cat. 115-035-003) or anti-rabbit (cat. 111-035-144) IgG secondary antibodies (Jackson ImmunoResearch, Cambridge, UK, dilution 1:3000) for 1.30 h at room temperature. The membranes were then washed again three times with PBST. Protein bands were visualized using the ECL chemiluminescence method (Clarity TM Western ECL Substrate, cat. 1705061, Bio-Rad Laboratories, CA, USA) and Chemidoc XRS (Biorad, Milan, Italy). Quantification of results was determined using ImageJ Software (version 1.52a, U.S. National Institutes of Health).

RNA Purification and Quantitative Real-Time PCR
Total RNA was extracted from cells using the QIAzol Lysis Reagent according to the manufacturer's instructions (cat. 79306, Qiagen, Hilden, Germany). Spectrophotometric quantization of each purified RNA was estimated using a Nanodrop apparatus (Thermo Fisher Scientific, MA, USA). The purified RNA was considered DNA and protein-free if the ratio between readings at 260/280 nm was ≥1.8. Isolated mRNA was reverse-transcribed using iScript Reverse Transcription Supermix for RT-qPCR (cat. 1708841, Bio-Rad, Italy). qPCR was carried out in a CFX96 real-time PCR detection system (Bio-Rad) with the use of SYBR Green master mix kit (cat. 1725271, Bio-Rad) and the following primers:

Flow Cytometry Analysis
Apoptosis was measured using the BD Pharmingen™ FITC Annexin V Apoptosis Detection Kit I (cat. 556547, BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's instructions. MDA-MB-231 cells were seeded (2.5 × 10 5 cells/well) in 35 mm culture dishes. The day next, the cells were either treated with erucin (30 µM) for 48 h or left untreated. MDA-MB-231 cells were later collected and stained with Annexin V-FITC/Propidium Iodide (PI). Flow cytometry analysis was performed using a BriCyte flow cytometer (Mindray, Italy). A minimum of 50,000 events for each sample were collected and data were analyzed using FlowJo v10 software (Tree Star, Ashland, OR USA).

Wound Healing Assay
MDA-MB-231 cells were seeded in 6-well plates (3 × 10 5 cells/well). Once the cells reached 90% of confluence, erucin 0.3 and 1 µM were added, and a wound area was created by scraping the cell monolayer with a sterile 200 µL pipette tip. Subsequently, the cells were incubated at 37 • C in 5% CO 2 . The width of the wounded area was photographed with an inverted microscope (20-fold magnification) at the time zero point as well as after 24 h and 48 h of treatment. The wounded area was measured using Image J software (version 1.52a, U.S. National Institutes of Health).

Clonogenic Assay
MDA-MB-231 cells were seeded in 6-well plates (1 × 10 3 cells/well) on the next day, treated with erucin (0.3 and 1 µM), and allowed to form colonies for 14 days. After washing with PBS, MDA-MB-231 colonies were fixed with 4% paraformaldehyde and colored with 0.5% crystal violet. Colonies composed of more than 50 cells were manually counted under the microscope and captured by a digital camera.

Invasion Assay
Boyden chambers with polycarbonate filters and a nominal pore size of 8 µm (cat. PIEP12R48, Millipore, USA) were coated on the upper side with Matrigel (Becton Dickinson Labware, USA). The chambers were placed in a 24-well plate and MDA-MB-231 cells (2.5 × 10 5 cells/mL) were plated in the upper chamber in the presence or absence of erucin (0.3 and 1 µM) in serum-free DMEM. At the end of the 16 h incubation period, the medium was removed, the filters were fixed with 4% formaldehyde for 2 min, and the cells were permeabilized with 100% methanol for 20 min. The methanol was removed and the chambers were stained with Giemsa for 15 min; they were then washed with PBS. The filters were removed and the nonmigrating cells on the top of the filter were removed using a cotton swab. The filters were then placed on a slide and examined under a microscope. Cell invasion was determined by counting the number of cells stained on each filter in at least 4-5 randomly selected fields.

Intracellular ROS Measurement
The generation of intracellular reactive oxygen species (ROS) was estimated using the fluorescence probe 2 ,7 -Dichlorfluorescein (DCF, cat. 35848, Sigma-Aldrich, Milan, Italy). For the experiments, MDA-MB-231 cells were plated in 96-multi-well black plates (Corning, USA) at a density of 10 4 cells/well and, after reaching 80% of confluence, were incubated with erucin 30 µM for 3 and 6 h. After washing with 100 µL/well of PBS, cells were incubated for 30 min with 200 µL of 10 µM DCF in HBSS containing 1% FBS. Finally, cells were washed and incubated with Fenton's reagent (H 2 O 2 /Fe 2+ 2 mM) for 3 h at 37 • C. The DCF fluorescence intensity was detected using a fluorescent microplate reader (excitation 485 nm and emission 538 nm; GloMax ® -Multi Detection System, Promega). The intracellular ROS levels were expressed as relative fluorescence intensity after normalizing to viable cell numbers. Additionally, the fluorescence images of differently treated cells were observed by fluorescence microscopy after washing twice with PBS. DCF was observed to emit green fluorescence.

Statistical Analysis
Data were expressed as mean ± SEM of n = 3 experiments. Data were analyzed and presented using GraphPad Prism 8.2.0 software (San Diego, CA, USA). Significance was determined using Student's 2-tailed t-test. Results were considered significant at p values less than 0.05 and are labelled with a single asterisk. In addition, p-values lower than 0.01 and 0.001 are indicated with double and triple asterisks, respectively.